The field of the invention relates to replication proteins and variants thereof which may be utilized in methods for assessing and modulating cell proliferation. In particular, the field of the invention relates to replication protein A 32 (“RPA32” otherwise referred to as “RPA2”), replication protein 4 (“RPA4”), variants thereof, polynucleotides encoding the polypeptides, and antisense polynucleotides thereof which may be utilized in methods for assessing and modulating cell proliferation.
The human replication protein A (RPA) is a trimeric complex found not only in human cells but also in other mammalian species and lower eukaryotes. RPA is a single-stranded DNA (ssDNA)-binding protein essential for DNA replication, repair, recombination, and monitoring cellular DNA-damage at cell cycle checkpoints. The three peptides of human RPA have molecular masses of approximately 70 kD (for the polypeptide called “RPA70” or alternatively “RPA1”); 32 kD (for the polypeptide called “RPA32” or alternatively “RPA2”); and 14 kD (for the polypeptide called “RPA14” or alternatively “RPA3”). All three polypeptides have been cloned and their encoding sequences have been reported (Erdile et al., 1991, J. BIOL. CHEM. 266:12090-12098; Erdile et al., 1990, J. Biol. Chem. 265:3177-3182; and Umbricht et al., 1993, J. BIOL. CHEM. 268:6131-6138).
RPA has been observed to exhibit functional activities associated with several characterized domains of the three RPA polypeptides. For example, RPA has been observed to bind ssDNA via several identified DNA-binding domains (DBDs) in the three polypeptides. RPA70 contains four domains: an N-terminal DBD-F domain and DNA binding domains (DBDs) A, B, and C. RPA32 contains three domains, including an N-terminal phosphorylation domain, a central DNA-binding domain (DBD-D), and a C-terminal winged-helix domain involved in protein-protein interactions. RPA14 includes a single domain referred to as DBD-E. Not all of the domains referred to as “DBDs” are involved directly in binding ssDNA. The N-terminal DBD-F domain of RPA70 is implicated in protein-protein interactions in DNA metabolism. The DBD-E domain (i.e., RPA14) has been shown to be structurally important for the formation of the RPA trimer.
In addition to RPA70, RPA32, and RPA14, human cells also have a homolog of RPA32, called RPA4. This subunit is expressed in colon mucosal and placental cells, and RPA4 is also detected in three of fourteen human cell lines examined (Keshav et al., “Rpa4, a Homolog of the 34-Kilodalton Subunit of the Replication Protein A Complex,” MOLEC. CELL. BIOL., June 1995, p. 3119-3128, Vol. 15, No. 6). RPA4 is able to interact with RPA14 and RPA70 to bind ssDNA.
It now has been shown that RPA4 can substitute for RPA32 with respect to some functions requiring RPA32 in human cells. Exogenous RPA4 expression can change the distribution of cells in S-phase caused by a RPA32 knockdown. Furthermore, RPA4 expression leads to an accumulation of cells in G2/M phase. In addition, a hybrid form of RPA32 containing the non-conserved basic region of RPA4 has properties like those of RPA4 in vivo. Recombinant polypeptides and polynucleotides that encode RPA4 or RPA32 variants, such as the disclosed RPA32 hybrids, may be expressed in cells in order to modulate proliferation. Also, expression of RPA4 has been observed to be reduced in proliferating or cancerous cells in comparison to normal cells.